PROJECT SUMMARY The genomes of higher organisms are highly annotated by specific chromosomal proteins and histone modifications along active genes, regulatory elements, or silent regions. An ongoing challenge is to decipher the rules that establish and maintain chromatin organization. In the upcoming funding period, we propose to focus on analysis of the Polycomb group (PcG) proteins, based on their central importance in development and disease, the intriguing hypotheses raised by our current results, and our ability to use new approaches to probe chromatin protein interactions with precision. Our studies will focus on the versatile Drosophila model, but due to the high conservation of this key regulatory system we can move between fly and human systems with ease. Our experiments will address two central problems in chromatin biology: understanding the targeting and spreading of chromatin domains, and probing the ability of chromatin factors to mark genes as future regulators of cell type specificity. In Aim 1, we will define the series of molecular interactions that coordinate canonical and variant PcG complexes. In Aim 2, we will analyze how H3K27me3 is propagated in cis, to create heritable repressive domains. In Aim 3, we will determine whether a bivalent state primes developmental genes in Drosophila. Success in our studies will be highly relevant to understanding normal and aberrant genome organization and function in development and disease.